Nicole Smoot. Biofuels are made by converting biomass into usable fuels (biodiesel and ethanol) Starches and sugars are fermented into bioethanol Currently,

Engineering Maize to More Efficiently Be Converted to EthanolNicole Smoot

Biofuels Today• Biofuels are made by

converting biomass into usable fuels (biodiesel and ethanol)

• Starches and sugars are fermented into bioethanol

• Currently, we mix 10% ethanol and 90% fossil fuels

• Although growing crops specifically for biofuels is an option, it uses a lot of land and doesn’t reduce green house gas emissions

• Instead, we can use agricultural or municipal waste

Cellulosic Ethanol: Current Method• Second generation biofuel (manufactured from

biomass)• Made by converting cellulose into ethanol• Current procedure:• 1) Pretreatment to separate cellulose from lignin• Lignin is a long polysaccharide that prevents the breakdown

and fermentation of cellulose. • 2) Cellulolytic Process breaks down cellulose• Cellulose is broken down either with chemicals or enzymes

• 3) Fermentation of the two resulting sugars, xylose and arabinose• 4) Distillation• 5) Dehydrating• 6) Denaturing

Maize (Zea mays)• Corn is one of the most abundant crops grown in

US (32% of the world’s corn), so there is a lot of potenial biofuel raw material.

• Already, most corn is genetically modified• Corn stover: leaves and stalks of maize. Usually,

stover is left in the fields after harvest. • Corn stover is high in cellulose, but also contains

about 20% lignin• Currently it is expensive and inefficient to break

down lignin.• Designed to protect the plant from being eaten by

bacteria, which makes it hard to degrade

Amycolatopsis• Amycolatopsis is a soil

bacteria found in Idaho• It is able to break down

lignin• Produces a catalase-

peroxidase enzyme called Amyco1that depolymerizes lignin

• Capable of surviving with only lignin as a source of carbon

• Specific strain to be used: Amycolatopsis sp. ATCC 39116 strain 75iv2

Intein Splicing• An intein is a section of amino acids in a protein

that can self-splice out• Inteins splice out at random times• Mutagenic PCR intentionally causes mutations in a

gene (approximately 2% will have a mutation)• A collection of slightly mutanized versions of a

gene is called a library. • By doing mutagenic PCR enough times on the

intein, an intein might be created that is induced to splice by cold.

Can we engineer corn cells to contain cold-activated

enzymes from Amycolatopsis that are

capable of breaking down the lignin in the cell wall?

WHYYYYYY• Corn• Cold• Lgingin• What is library• What is intein• Not out of dna out of protein• Way of testing if intein is functonslal• Why is rfp• Agro get in how not liq rub wound• What mpcr• Da f is da FLAG for?

Overview of Method1) Create an intein

library and insert into GFP gene. Put into

Agrobacterium tumefaciens and

cultivate on plates.

2) Isolate colonies that begin to glow when exposed to cold.

3) Cut out intein nucleotide sequence from plasmid and put

into a gene for Amyco1 in a new vector with RFP.

Insert new vector into new Agro cells.

5) Inoculate maize plants with Agro. Test to see if the Amyco1 enzyme is functional

only when the plant is frozen

4) Cultivate Agro cells in a liquid buffer with corn stalk. Test to see if freezing the culture results in decreased

lignin.

GFP Vector Preparation• Perform mutagenic PCR on the intein (Clontech)

and insert into a GFP (green fluorescent protein) gene (LifeTechnologies).

• Insert the GFP gene into a Ti plasmid (tumor-promoting and opine-synthesis genes removed) from Agrobacterium tumefaciens (Clontech).

• Replace the plasmid inside the Agrobacterium tumefaciens and culture in broth for one day.

Locating Cold-Regulated Intein• Plate the Agrobacterium. Incubate at 20°C for

three days. • Check colonies under black light to see if any

fluoresce. • Let plates sit at 0°C for six hours. Return to 20°C

and let sit for 12 hours. • Check plates under black light again to see if any

have started to fluoresce.• Isolate any candidate colonies and culture in

liquid broth (one tube per colony) for two days. • Plate ten plates per tube. Place at different

temperatures (0°C, 2°C… 20°C) for six hours. Check each plate under black light before and after.

Variables/ControlsVariable ControlGFP gene with mutanized intein in Agro

GFP gene with normal intein in Agro

GFP without intein in Agro

Agro with candidate intein in GFP at 0°C, 2°C… 18°C

Agro with candidate intein in GFP at 20°C

Agro with no intein in GFP at 0°C, 2°C…20°C

Preparing Vector with Amyco1• Cut out intein using the same restriction enzymes

used when creating the GFP gene. • Use restriction enzymes to place chosen intein

gene sequence in the gene for peroxidase Amyco1 (ATCC). Insert gene for Amyco1 into a Ti plasmid (Clontech), using 35S promoter. Add the cell wall targeting signal sequence of barley alpha amylase to the 5’ end of the Amyco1 gene (Megazyme).

• The targeting signal sequence tells the cell to send the Amyco1 enzyme to the cell wall.

Preparing Vector with Amyco1 (cont)• Attach a FLAG epitope to the Amyco1 gene for

future Westerns.• On a separate promoter (TK promoter), insert the

gene for RFP (LifeTechnologies) to check if the plant was inoculated effectively.

• Insert the plasmid into new Agrobacterium tumefaciens cells.

• Culture cells on plates, then transfer colonies to liquid broth.

Testing Amyco1• Test concentration of lignin in a piece of corn leaf

using UV-spectrophotometric analysis.• UV spectrophotometric analysis is done by shining

a laser into a substance. Based on how the light is absorbed, the size and quantity of molecules in the solution can be defined.

• Place a piece corn leaf in a tube and add transformed Agrobacterium.

• Incubate at 20°C for a day, then freeze for six hours. Return to 20°C and incubate for a week.

• Test lignin concentration of corn leaf again using UV-spectrophotometric analysis.

Expected ResultsTest Expected Outcome

Corn leaf (with Agro), 0°C

Decreased Lignin

Corn leaf (No Agro), 0°C and 20°C

Normal Lignin

Corn leaf (with Agro), 20°C

Normal Lignin

Production of Transgenic Maize Plants• Transform immature zygotic maize embryos using

Agrobacterium tumefaciens. • Use RFP reporter gene to ensure the plants were

effectively inoculated. • Grow embryos for 10-12 weeks. • Run a Western blot test on normal leaves as well

as leaves that were frozen for 4 to 6 hours then returned to 20°C for 12 hours. Use anti-FLAG antibodies (Sigma-Aldrich) to identify the Amyco1 enzyme.

• Do UV-spectrophotometric analysis of both normal leaves and frozen and thawed leaves to test lignin concentration.

Expected ResultsTest Expected OutcomeFrozen corn leaves (with Agro)

Lower lignin concentration

Frozen corn leaves (no Agro)

Normal lignin

Normal corn leaves (no Agro)

Normal lignin

Normal corn leaves (Agro)

Normal lignin

Potential Complications• Possibly no cold-regulated inteins produced• Cold-regulated intein may only work in GFP• DDUN FORGET DIS LKGJLKSD diff btwn cold n

chance may confuse bc we dk if it is just doing da splicing randomly or actually bc of cold yo

• Amyco1 enzyme might not be functional in maize• The cell wall targeting sequence may not attack

the cell wall in maize

Implications for Biofuel Production• Could make cellulosic ethanol as inexpensive as

corn ethanol without competing with food for land• This process could be transferred to other plants

that can grow in marginal land (ex. some grass species)

• Biofuels could become more widespread and decrease our dependence on fossil fuels

Acknowledgements• Special thanks to all our instructors, Dr. Paul

Feldstein, Dr. LeAnn Lindsay, Dr. Adam Telleen, Ann Moriarty, and Dana de Farcy for answering all of my questions.

• Thanks to all the authors I cited in this presentation.

• Thanks to the Cluster 1 fam, you guys are the best.

Works Cited• Bugg, Timothy et al. "The Emerging Role for Bacteria in Lignin Degradation and Bio-

product Formation." The Emerging Role for Bacteria in Lignin Degradation and Bio-product Formation. ScienceDirect, 9 Nov. 2010. Web. 29 July 2015. http://www.sciencedirect.com/science/article/pii/S0958166910001977

• Shen, Binzhang et al. "Engineering a Thermoregulated Intein-modified Xylanase into Maize for Consolidated Lignocellulosic Biomass Processing." Nature Biotechnology, 31 May 2012. Web. 29 July 2015. http://www.nature.com/nbt/journal/v30/n11/full/nbt.2402.html#methods

• Frame, B et al. “Genetic Transformation using maize immature zygotic embryos." National Center for Biotechnology Information. U.S. National Library of Medicine, 2011. Web. 29 July 2015. http://www.ncbi.nlm.nih.gov/pubmed/21207278

• Das, A., and S. Stachel. "Promoters of Agrobacterium Tumefaciens Ti-plasmid Virulence Genes." Nucleic Acids Research. US Library of Medicine, 1986. Web. 29 July 2015. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC339509/pdf/nar00272-0225.pdf

• Zahm, P., SL Rhim, and K. Geider. "Promoter Activity and Expression of Sequences from Ti-plasmid Stably Maintained in Mammalian Cells."National Center for Biotechnology Information. U.S. National Library of Medicine, 1989. Web. 29 July 2015. <http://www.ncbi.nlm.nih.gov/pubmed/2481809>

Picture Creds• http://www.fotosearch.com/CSP196/k1963077/• https://www.flickr.com/photos/hand_rail/7341983892• http://schoolworkhelper.net/biofuels-effect-on-social-eco

nomic-political-environmental/

• http://www.greenoptimistic.com/us-department-of-energy-plans-for-the-future-with-biofuel-projects-20121201/

• https://upload.wikimedia.org/wikipedia/commons/9/95/Schematic_of_UV-_visible_spectrophotometer.png

• http://collegehillfarmmarket.org/2015/07/07/corn-sweet/• http://

wwnorton.com/college/biology/micrograph/single.aspx?fig=176

• http://www.mdpi.com/1422-0067/14/4/6960